The proper orchestration of the steps required for orderly progression of the cell through the cell cycle requires a number of signaling pathways within cells. Many of these pathways utilize protein kinases to effect the transmission of crucial signals at the appropriate time and intracellular location. Cell cycle kinases are naturally occurring enzymes involved in regulation of the cell cycle which is generally divided into four segments: G1 (gap1), S (DNA systhesis), G2 (gap 2) and M (mitosis). Some of these kinases are responsible for inhibiting the cell's normal progression through cell division, while others are normally active in promoting the progression of cells through the cell cycle leading to cell division. Increased activity or temporally abnormal activation of these kinases has been shown to result in development of tumors and other proliferative disorders.
One of the protein kinases involved is a tyrosine specific kinase, Wee1, that has as its substrate another kinase complex called Cdc2/cyclinB. Wee1 kinase is a regulatory kinase that has Cdc2/cyclinB as its substrate and when Wee1 is active, it phosphorylates a specific tyrosine (Tyr15) on Cdc2 that causes an inactivation of the Cdc2/cyclinB complex which in turn results in a pause or checkpoint in the cell cycle at the G2 and M transition. The kinase activity of Cdc2/cyclinB is absolutely required for cells to progress through the G2 stage of the cell division cycle to the M (or mitotic) phase where two daughter cells are formed from the division of the parent cell. Under normal circumstances, as cells are progressing through the cell cycle, the Cdc2/cyclinB complex is assembled in late S phase and through G2. Normally, Wee1 is active and thus phosphorylates the Cdc2/cyclinB complex until the end of G2 when all of the necessary components have been synthesized for the entry of cells into M phase. Wee1 activity then diminishes, a phosphatase removes the inhibitory phosphorylation from Tyr15 of Cdc2/cyclinB, the complex becomes activated and cells move into M phase where the replicated DNA is divided and the daughter cells are formed. Inhibition of Wee1 results in no inhibitory phosphorylation of Tyr15 on Cdc2/cyclinB and the potentially inappropriate and premature entry of the cell into mitosis.
In addition to regulation of the transition of cells between the different phases of the cell cycle under normal conditions, the cell cycle transitions are regulated in response to damage to DNA presumably giving cells opportunities either to repair potentially genotoxic DNA damage before replication using a damaged DNA template or to permanently exit the cell cycle and die.
Another kinase of interest named Chk1 participates in this DNA damage dependent signaling pathway by phosphorylating a phosphatase called Cdc25C which when itself is active and co-localized with Cdc2/cyclinB in the nucleus, dephosphorylates Tyr15 and causes the activation of the Cdc2/cyclinB complex. The Chk1 mediated phosphorylation of Cdc25C causes Cdc25C to be exported from the nucleus at which point it is no longer able to dephosphorylate and thus activate Cdc2/cyclinB. Therefore, if Chk1 is active (in response to DNA damage) it will indirectly contribute to the inactivation of Cdc2/cyclinB (whose activity is required for progression into M phase) through the preservation of the inactivating phosphorylation of Tyr15 on Cdc2/cyclinB. Conversely, inhibition of Chk1 would result in the dephosphorylation of Cdc2/cyclinB by the phosphatase Cdc25C in the nucleus (not exported to the cytoplasm since it is not phosphorylated by Chk1) and the consequent activation of Cdc2/cyclinB with the accompanying entry of the cells in mitosis.
Inhibition of Wee1 or Chk1 or both kinases in the presence of DNA damaged by conventional DNA-directed chemotherapeutic agents or by radiation presents an opportunity to utilize cellular regulatory pathways to inappropriately and prematurely cause cells to progress into M phase. Such cells may be less likely to survive and further divide since the commitment to M phase was made in the presence of potentially catastrophically damaged DNA (Alan J. Kraker and Robert N. Booher, “New Cell Cycle Targets,”, Ann. Rep. Med. Chem., 1999; 34:247–256).
Small molecule inhibitors of Wee1 kinase have been reported, WO 0119825 and Cancer Res. (2001), 61(22), 8211–8217. Small molecule inhibitors of Chk1 kinase have also been reported W00016781, Cancer Res. (2000), 60(3), 566–572.
Pyrrolocarbazole derivatives are known to have inhibitory activity against Protein kinase c and anti tumor activity (U.S. Pat. No. 4,912,107) but compared to the compounds of the present invention, the compounds disclosed in U.S. Pat. No. 4,912,107 have very low checkpoint kinase abrogator activity. Pyrrolocarbazole derivatives are also known to stimulate platelet production (WO96/28447) and to promote thrombopoiesis (WO9809967). EP 0695755 discloses another pyrrolocarbazole derivative having Protein kinase c activity. U.S. Pat. No. 5,166,204 discloses antitumor isoindole derivatives having a linkage or lower alkylene group bonded to the 2 and 3 or 3 and 4 of a carbazole skeleton. U.S. Pat. No. 5,728,709 discloses pyrrolocarbazole derivatives that stimulate platelet production. WO01/85686 also discloses pyrrolocarbazole derivatives.
However, there are no reports that any type of pyrrolocarbazole inhibits either Wee1 kinase or Chk1 kinase. Nor have there been any reports that any type of pyrrolocarbazole inhibits both Wee1 kinase and Chk1 kinase.